Fusion of the mixed lineage leukemia protein (MLL) to one of over 50 different translocation partners converts it into a potent leukemogenic oncoprotein. The resulting fusion proteins transform by upregulation of A cluster Hox genes including Hoxa9 and the Hox cofactor Meis1. We have made considerable progress toward understanding the mechanism of transcriptional regulation by MLL and MLL fusion proteins. We discovered that MLL is a histone H3 lysine 4 methyltransferase that regulates transcription by direct binding to both Hox and Meis1 promoters and coding regions. We found that MLL interacts with the tumor suppressor menin via a small amino terminal domain and that this interaction is important for both the function of normal MLL as well as for MLL fusion proteins. Our overriding goal is to facilitate the development of more effective therapies for leukemias with MLL rearrangements. To this end, the proposed experiments will analyze several mechanisms pivotal for transformation by MLL fusion proteins. Some MLL fusion proteins fuse MLL to transcriptional activators while others dimerize the truncated MLL molecule. Both classes share the MLL amino terminus, which is required for transformation. In SA#1 we will analyze the role of the interaction of the MLL amino terminus with menin in transformation by both classes of MLL fusion proteins. SA#2 focuses on the most common MLL fusion proteins, involving translocation partners AF4, ENL, AF9 and AF5q31, which account for more than 70% of MLL-associated leukemias. All four proteins are physically associated in a complex called MPAC (MLL Partner Activation Complex) that includes Dot1, a histone H3 lysine 79 methyltransferase, and CDK9/CyclinT1/T2, which phosphorylates the C terminal domain of RNA polymerase II. We will study the mechanism of transcriptional activation by MPAC in both normal hematopoiesis and when recruited to target loci by leukemogenic MLL fusion proteins. Surprisingly, MPAC also contains Pc3 and Ring1B, two members of the repressive Polycomb group protein family. We will determine if this enzymatic activity of these proteins is intact in the context of MPAC and whether MLL fusion proteins prevent Polycomb-mediated silencing. SA#3 assesses the role of MPAC kinase and histone methyltransferase activity on fusion protein-mediated transformation. Studies of these potential "Achilles heels" will provide valuable insights into how normal transcriptional mechanisms are disrupted by MLL fusion proteins and how these may be targeted therapeutically.